登革病毒实时荧光PCR快速检测技术研究

Study on the rapid detection of dengue viruses by Taqman MGB real-time fluorescent PCR

目的建立TaqmanMGB实时荧光PCR技术快速检测登革病毒。方法利用TaqmanMGB技术，根据登革病毒3’端非编码区的一段高度保守序列，设计登革1～4型荧光PCR通用引物和TaqmanMGB探针，以登革热毒株作为标准，以乙脑毒株作对照，建立实时荧光PCR检测登革病毒的快速方法。并对8份ELISA法检测阳性的临床血清标本进行RT-PCR及荧光PCR扩增。结果RT-PCR检测8份临床血清标本，2份阳性，阳性率为25％。实时荧光PCR检测登革病毒cDNA灵敏度为0．17pg／μl，病毒液灵敏度为10^-5TCID50与乙脑病毒无交叉反应，检测8份临床血清标本，5份阳性，阳性率为62．5％。从RNA提取到检测结果仅需4h。结论TaqmanMGB实时PCR检测方法快速、敏感性高、特异性强，可作为登革病毒的快速检测方法，应用于登革热的临床早期诊断。

Objectlve To develop the Taqman MGB real-time fluorescent PCR assay for rapid detection of Dengue viruses. Methods Using Taqman MGB technique, a pair of universal primers and Taqman MGB probe were designed according to a highly reserved sequence of the 3＇-noncoding region of dengue viruses type 1-4. Dengue virus strains were used as standard and Japanese encephalitis virus strains were used as control, the real-time PCR assay for specific and sensitive detection of the dengue viruses was established. While 8 serum specimens of ELISA positive were detected by the RT-PCR and fluorescent PCR. Results The sensitivity of real-time PCR was 0.17pg/μl（eDNA）or 10 5TCIDso. There was no cross-reaction with Japanese encephalitis virus. Of 8 specimens, 2 were positive by RT-PCR and 5 were positive by real-time PCR. The test could be completed in 4 hours. Conelusion The Taqman MGB real-time PCR assay was fast, sensitive and specific. It could be applied to the quick early diagnosis of dengue viruses.